Deletions in the third intracellular loop of the thyrotropin receptor. A new mechanism for constitutive activation

Gain-of-function mutations of the thyrotropin receptor (TSHR) gene have been invoked as one of the major causes of toxic thyroid adenomas. In a toxic thyroid nodule, we recently identified a 9-amino acid deletion (amino acid positions 613-621) within the third intracellular (i3) loop of the TSHR resulting in constitutive receptor activity. This finding exemplifies a new mechanism of TSHR activation and raises new questions concerning the function of the i3 loop. Because the i3 loop is thought to be critical for receptor/G protein interaction in many receptors, we systematically reexamined the role of the TSHR's i3 loop for G protein coupling. Thus, various deletion mutants were generated and functionally characterized. We identified an optimal deletion length responsible for constitutive activity. If the number of deleted amino acids was reduced, elevated basal cAMP accumulation was found to be concomitantly diminished. Expansion of the deletion dramatically impaired cell surface expression of the receptor. Shifting the deletion toward the N terminus of the i3 loop resulted in unaltered strong constitutive receptor activity. In contrast, translocation of the deletion toward the C terminus led to significantly reduced basal cAMP formation, most probably due to destruction of a conserved cluster of amino acids. In this study, we show for the first time that amino acid deletions within the i3 loop of a G protein-coupled receptor result in constitutive receptor activity. In the TSHR, 75% of the i3 loop generally assumed to play an essential role in G protein coupling can be deleted without rendering the mutant receptor unresponsive to thyrotropin. These findings support a novel model explaining the molecular events accompanying receptor activation by agonist.